Identification of Metabolic Vulnerabilities of Ras-Driven Cancer Cells Tumor growth requires biosynthesis of protein, DNA, RNA, and membrane. Cellular metabolism provides the substrates and energy for this biosynthesis. Consistent with the central role of metabolism in cancer growth, altered metabolism is a hallmark of cancer. The best-known example is avid glucose fermentation even in the presence of oxygen, i.e., the Warburg effect. Recently, it has become clear that a major function of oncogenes is to induce metabolic changes, including the Warburg effect, to provide substrates and energy for biosynthesis that enables tumor growth. Thus cancer cells are vulnerable to interference in metabolic pathways, hence the need to target metabolism for cancer therapy. Ras genes are among the most frequently mutated oncogenes in cancer, and their mutational activation induces the Warburg effect. Unlike for many oncogenic signaling proteins, there are no safe and effective pharmacological inhibitors of activated Ras, increasing the importance of understanding metabolic vulnerabilities of Ras-driven tumors. We have recently examined the metabolic consequences of Ras activation in tumor cells via metabolomics and isotope-tracer studies. This revealed that Ras not only induces aerobic glycolysis, but also decreases acetyl-CoA production from glucose and fatty acids, and enhances dependence of the TCA cycle on glutamine, revealing a metabolic vulnerability. Moreover, we found that Ras activates the catabolic cellular self-digestion process of autophagy, that autophagy sustains TCA cycle metabolism, and that tumor cells with activated Ras are dependent on autophagy for survival and tumorigenesis. Thus, activated Ras leads to autophagy addiction, revealing another metabolic vulnerability. Our unifying hypothesis is that Ras decreases input into the TCA cycle from glucose and fatty acids, creating the requirement for glutamine and other autophagy-supplied TCA cycle substrates to sustain tumor cell metabolism. Here we aim to test this hypothesis and understand more comprehensively the underlying mechanisms, generality of the metabolic alterations induced by Ras and the best ways to exploit them. To this end we will combine state-of-the-art metabolomics with in vitro and in vivo cancer models driven by Ras or the downstream kinases Akt and Raf. The net effect of this research will be to dramatically increase understanding of the interplay of oncogene signaling and metabolism, and in so doing to identify new therapeutic targets for Ras-driven cancers. This project is a direct extension of the very productive collaboration between the laboratories of Dr. Eileen White (Rutgers University) and Dr. Josh Rabinowitz (Princeton University) previously funded by a NIH Challenge Grant on cancer metabolism. PUBLIC HEALTH RELEVANCE: We have known for over 50 years that a major feature that distinguishes normal cells from cancer cells is altered metabolism. Only recently has it become clear that activation of oncogenes and loss of tumor suppressor genes reprograms metabolism to generate the building blocks for production of new tumor cells and to meet the energy requirements for cancer cell growth. Oncogenic forms of Ras dramatically alter cellular metabolism, promote tumorigenesis and are associated with poor prognosis. Targeting Ras therapeutically has been difficult, necessitating approaches to block pathways, such as metabolic pathways, downstream of Ras. We propose to use state-of-the-art mass spectrometry and cancer models to determine how Ras alters metabolism. This will expose vulnerabilities that can be exploited in the development of new cancer therapies.